Using transgenic zebrafish that overexpress Myc, a central regulator of human T-cell acute lymphoblastic leukemia (T-ALL), we developed a zebrafish model of T-ALL with features that closely resemble the human disease. Here we propose to conduct the first modifier screen in a vertebrate system to identify both enhancers and suppressors of T-ALL in the zebrafish. We predict that enhancers of the T-ALL phenotype will represent mutations in genes that have tumor suppressor characteristics and that suppressors will be mutations in parallel pathways that inactivate genes required for transformation and thus encode candidate drug targets. The central hypothesis for this research is that mutations will be identified in zebrafish that accelerate or delay the onset of Myc-induced T-ALL, and that the roles of the genes affected by these mutations will be conserved in human T-ALL pathogehesis. One immediate goal will identify genes directly or indirectly involved in the molecular pathways of malignant transformation induced by Myc, lending insight into the molecular basis for T-ALL pathogenesis. An important long-term goal is to find potential gene targets and molecular pathways for the development of more specific and less toxic drugs for the treatment of T-ALL. These goals will be achieved through three specific aims. In Aims 1 and 2, we will perform a genetic modifier screen to identify and clone enhancer and suppressor mutations that affect the rate of onset of Myc-induced T-ALL. Since the zebrafish is transparent, the onset of leukemia can be observed as GFP-labeled T cells expand beyond the region of the thymus. During the previous grant cycle, we created separate transgenic zebrafish lines expressing TAL1, LM02, and EGFP-mMyc in T cells and have isolated a p53 mutant zebrafish line that is tumor prone. In Aim 1, we will also generate separate transgenic zebrafish lines with T-cell-specific expression of HOX11, HOX11L2, TAN1-EGFP, and mutated NOTCH11. We will test these transgenic and mutant lines, in addition to a panel of 315 retroviral insertional zebrafish lines (representing inactivating mutations in identified genes) generated by Dr Nancy Hopkins'lab, for modifiers of m/Wyc-induced onset of T-ALL. To achieve a more extensive coverage of the genome as well as to expand the spectrum of genomic mutational alterations, we propose in Aim 2 to perform an ENU-based Myc modifier screen, which will involve the random introduction of point mutations at a rate of approximately 100 inactivating mutations per genome. We have made major progress during the previous grant cycle in defining at least 5 multistep oncogenic pathways in T-ALL of children and adults. In Aim 3, we will determine the clinical consequences of these gene expression programs in childhood T-ALL and identify new proteins that can be targeted to develop highly specific therapies. The results should provide a new rationale for subclassifying patient groups at diagnosis, especially high-risk patients who may benefit from alternative therapies. Aim 3 will raise numerous opportunities to interact closely with other projects in this program with the ultimate goal of bringing novel targeted therapies to the bedside for children and adults with T-ALL. We are confident that the research outlined here will lead to the discovery of critical genes in the molecular pathways that drive T-ALL, which may provide novel targets for T-cell leukemia-specific therapies.